Rhizobium-legume symbiosis has been recently reviewed by D. P. S. Verma and S. Long (1983) Intnatl. Rev. Cytol. Suppl. 14:211-245. J. E. Beringer (1982) in Proc. 8.sup.th N. Amer. Rhizobium Conf., eds.: K. W. Clark and J. H. G. Stephens, review transfer of symbiosis plasmids (pSym) between fast-growing species of Rhizobium. Fast-growing rhizobia (e.g. R. leguminosarum, R. meliloti, R. phaseoli, and R. trifolii) are classified within the family Rhizobiaceae. Following are representative examples of publications disclosing transfer of symbiosis plasmids and genes.
S. Higashi (1967) Gen. Appl. Microbiol. 13:391-403, transferred the ability to nodulate (i.e. Nod phenotype, or nod gene) clover from R. trifolii to R. phaseoli without artificially genetically marking the transferred pSym. A. W. B. Johnston et al. (1978) Nature 276:634-636, transferred a Tn5-marked conjugative plasmid that directed synthesis of a bacteriocin from a pea-nodulating R. leguminosarum into clover-nodulating R. trifolii and kidney bean-nodulating R. phaseoli. The resultant transconjugants were observed to gain the ability to form nodules on peas, though the ability to fix (i.e. Fix phenotype) nitrogen was not the same for all transconjugants. Conversely, P. J. J. Hooykaas et al. (1981) Nature 291:351-353, similarly transferred a Tn5-marked conjugative plasmid from R. trifolii to R. leguminosarum, resulting in effective nodulation on clover, and to Agrobacterium tumefaciens (a member of the family Rhizobiaceae), producing abaerrant, Fix.sup.- clover nodules. B. G. Rolfe et al. (1983) in Molec. Genet. Bacteria-Plant Interactions, ed: A. Puhler, pp. 188-203, report a R. leguminoserum-derived Sym plasmid that exchanged pea-specific functions for clover-specific functions by in vivo recombinational exchange of 60 kilobase pairs (kbp) of R. trifolii pSym DNA. The resultant transconjugants were Nod.sup.+ Fix.sup.+ on clover. J. W. Lamb et al. (1982) Mol. Gen. Genet. 186:449-452, found that a R. phaseoli plasmid could confer to R. leguminosarum the ability to nodulate and fix nitrogen on beans.
N. J. Brewin et al. (1980) J. Gen. Microbiol. 116:261-270, demonstrated that the plasmid of Johnston et al., supra, could complement nod.sup.- and fixation-defective (fix.sup.- gene) mutants of R. leguminosarum and restore strains to Nod.sup.+ and Fix.sup.+ phenotypes. N. J. Brewin et al. (1980) J. Gen. Microbiol. 120:413-420 showed that transfer of conjugative plasmids between different strains of R. leguminosarum can change the host-range specificity for effective symbiosis of the Rhizobia for different varieties of peas. G. Hombrecher et al. (1984) Mol. Gen. Genet. 194:293-298, found that the R. leguminosarum gene(s) determining which pea varieties are nodulated are located within a 5 kbp region of DNA. A. Kondorosi et al. (1983) in Puhluer, supra, pp. 56-63, have tentatively identified a particular nod gene determining host specificity in R. meliloti.
Z. Banfalvi et al. (1983) Mol. Gen. Genet. 189:129-135, have made R-prime plasmids carrying symbiotic genes of R. meliloti. A. Kondorosi et al. (1982) Mol. Gen. Genet. 188:433-439, introduced a RP4 mobilization (mob) site into a R. meliloti pSym and in the presence of RP4 and related plasmids were able to transfer the resultant plasmid and Nod.sup.+ Fix.sup.+ phenotypes into other rhizobial stains. J. A. Downie et al. (1983) Mol. Gen. Genet. 190:359-365, and S. R. Long et al. (1982) Nature 298:485-488, have made recombinant DNA plasmids in vitro which carry nod genes of R. leguminosarum and R. meliloti, respectively, that are capable of complementing nod- mutants. The R. leguminosarum genes were located on a 10 kbp DNA region that, when transferred into a R. phaseoli strain cured of its endogenous pSym, caused nodulation of peas. E. Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452, have identified nod and fix gene-containing clones in a cosmid library of R. meliloti. Hombrecher et al., supra. have identified nod gene-carrying cosmids derived from R. leguminosarum.
Replacement of absent (e.g. deleted) or defective (e.g. mutated) genes is a technique well known in the art of microbiology. Replacement of nod and fix is described in a number of publications, including, but not limited to, the following above cited works: Johnston et al., Hooykaas et al., Rolfe et al., Lamb et al., Brewin et al., J. Gen. Microbiol. 116:261-270 and 120:413-420, Hombrecher et al. A. Kondorosi et al. (1983) and (1982), Banfalvi et al., Downie et al., Long et al., and E. Kondorosi et al. Replacement of a gene generally entails introduction of a plasmid carrying a functional copy of that gene into a cell. Often the cell is previously cured of an endogenous plasmid carrying that gene.
J. O. Berry and A. G. Atherly (1982) in Proc. 8th N. Amer. Rhizobium Conf., eds: K. W. Clark and J. H. G. Stephens, pp. 115-128, and (1984) J. Bacteriol. 157:218-224, introduced plasmids into slow-growing strains of R. japonicum but did not demonstrate conjugal transfer of Glycine-active symbiotic genes between bacterial strains. R. lupini, various Rhizobium "species", and most strains of R. japonicum are slow-growers. K. S. Engwall and A. G. Atherly (March 1984) Abstracts, Amer. Soc. Microbiol. Annual Meeting, St. Louis, MO, p. 111, and K. S. Engwall et al. (1984) in Adv. Nitroqen Fixation Res., eds: C. Veeger and W. E. Newton, p. 679, have presented data indicating that Agrobacterium tumefaciens cells containing plasmid from a fast-growing R. japonicum strain will form associations with soybean roots, the roots then forming "bumps" reminiscent of root nodules (i.e. aberrant nodules) but lacking bacteroid release and ineffective in nitrogen fixation. A large plasmid present in USDA191, designated herein as pSym 191, had DNA sequences homologous to nitrogen fixation (nif) genes of Klebsiella pneumoniae (E. R. Appelbaum et al., in Veeger and Newton, supra, pg. 670) and R. meliloti (R. V. Masterson et al. (1982) J. Bacteriol. 152:928-931) as determined by nucleic acid cross-hybridization. It is believed that the present invention is the first disclosure of transfer between different Rhizobium strains of genes determining host specificity of the symbiosis of rhizobia with any members of the genus Glycine and in particular soybean (Glycine max L.) varieties.
Plasmid vectors that can be transferred from E. coli to Rhizobium, that are maintainable in E. coli but not in Rhizobium, and that carry a mob-bearing transposon capable of mobilizing DNA transfer among a wide range of gram-negative bacteria have been disclosed by R. Simon et al. (1983) in Molec. Genet. Bacteria-Plant Interactions, ed. A. Puhler, pp. 98-106. Such vectors are of a class known as "suicide vectors". No cells can be stably transformed by autonomous suicide vector; the vector, having a replicon not capable of being maintained in rhizobia, "commits suicide" after introduction into a rhizobial cell. After cells have been transformed by a suicide vector, the only cells isolated after selection for a transposon-carried genetic marker will be those having a copy of the transposon incorporated into a replicon-containing DNA molecule already present and stably maintained in the cell.
The combination of rhizobial cells with a carrier material thereby forming an inoculant composition or a seed coating composition, and additionally with a binder material thereby forming a seed coating composition, is a well understood technology. The use of such compositions is also well understood.